In spite of considerable effort, the identities of the exact covalent modifications to lens proteins that accompany the process of human senile cataractogenesis have remained largely unknown. Identification of such modifications will greatly increase our understanding of the basic biochemical mechanisms involved in human cataractogenesis, and will provide a rational basis for the future development of pharmacological agents to treat this disorder. During the last grant period, we have used antisera made against synthetic peptides to quantitatively probe Western blots of proteins from opaque versus transparent tissue from the human lens, to demonstrate covalent changes in a number of lens polypeptides, which allows us to use anti-peptide sera to identify and purify tryptic peptides that contain the covalently modified sequences. In the next grant period, we propose to use the methodology to identify and purify tryptic peptides from lens proteins, that contain amino acid sequences that have been covalently modified during the process of human senile cataractogenesis. These tryptic peptides will be further characterized by mass spectral analysis, to determine which the amino acids have been modified, as well as to determine the chemical nature of the modification. Using the same methodology, we will analyze opaque versus transparent lenses of the Emory mouse strain, to determine the time course of pre-cataractous changes occurring in these lenses. Thus, from a single human or animal cataractous lens, we will be able to screen through the thousands of possible tryptic peptides generated from total lens proteins, to identify and purify those that are modified during cataractogenesis, so that the exact chemical nature of these cataract- related modifications can be identified.